The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia.
The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia.
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DOI：
10.1042/bj20101957
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发表时间：
2011-06-01
期刊：
影响因子：
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通讯作者：
Halestrap AP
中科院分区：
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作者：
Pasdois P;Parker JE;Griffiths EJ;Halestrap AP
氧化细胞色素c是线粒体膜间隙内一种强大的超氧化物清除剂，但这种作用在原位的重要性尚未得到很好的探讨。在本研究中，我们研究了这一点，并特别强调了心肌缺血期间线粒体细胞色素c的损失是否可能介导随后再灌注过程中ROS（活性氧）的产生增加，从而诱导mPTP（线粒体通透性过渡孔）打开。用洋地黄苷或缺血30min诱导大鼠心肌线粒体细胞色素c耗竭。对照和细胞色素c缺陷线粒体与混合呼吸底物和adp再生系统（状态3.5）孵育以模拟生理条件。这与大多数发表的使用单一底物且没有显著ATP转换的研究形成对比。缺乏细胞色素c的线粒体比对照线粒体产生更多的H2O2，外源细胞色素c的加入逆转了这种增加。在缺血前和缺血末线粒体产生H2O2的[KCN]速率增加的情况下，与氧化细胞色素c含量相关，但与呼吸或NAD(P)H自身荧光速率无关。缺血期间细胞色素c的丢失不是由mPTP开放介导的（环孢素a不敏感），也与线粒体Bax、Bad、Bak或Bid的变化无关。然而，结合的HK2（己糖激酶2）和Bcl-xL在缺血终端线粒体中降低。我们得出结论，在病理生理条件下，由外膜渗透引起的缺血期间细胞色素c的损失是线粒体产生H2O2的主要决定因素。我们进一步提出，在缺氧条件下，H2O2的产生激活信号通路也可能是由氧化细胞色素c的减少和超氧化物清除的减少介导的。
Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H2O2 to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging.